The effect of the anti-coagulant EDTA on the deposition and adhesion of whole blood deposits on non-porous substrates.

An investigation into whether the addition of a commonly used anti-coagulant agent like ethylenediaminetetraacetic acid (EDTA) has an impact on the adhesion potential of blood to non-porous substrates was conducted. Two non-porous substrates (aluminum and polypropylene) exhibiting six different surface roughness categories (R1-R6) were used as test substrates upon which either whole blood or blood treated with EDTA was deposited. Samples were exposed to different drying periods (24 hours, 48 hours, and 1 week) before undergoing a tapping agitation experiment in order to evaluate the adhesion to the surface. Clear differences in adhesion potential were observed between whole blood and blood treated with EDTA. Blood treated with EDTA displayed a stronger adhesion strength to aluminum after a drying time of 24 h pre-agitation, while whole blood presented with a stronger adhesion strength at the drying time of 48 h and 1 week. Both EDTA-treated and EDTA-untreated blood was shown to dislodge less easily on polypropylene with the only difference observed on smooth surfaces (0.51-1.50 µm surface roughness). Thus, when conducting transfer studies using smooth hydrophobic substrates like polypropylene or considering the likelihood of transfer given specific case scenarios, differences in adhesion strength of blood due to hydrophobic substrate characteristics and a decreased surface area need to be considered. Overall, whole blood displayed a better adhesion strength to aluminum, emphasizing that indirect transfer probability experiments using EDTA blood on substrates like aluminum should take an increased dislodgment tendency into account in their transfer estimations.

The impact of substrate characteristics on the collection and persistence of biological materials, and their implications for forensic casework.

This study assessed the level of nucleic acid persistence on the substrate pre-, and post-swabbing, in order to assess whether biological materials (touch, saliva, semen, and blood) are collected differently depending on the substrate characteristics. A total of 48 samples per deposit and substrate variety (n = 384) were assessed by tracking the persistence of nucleic acid using Diamond™ Nucleic Acid Dye (DD) staining and Polilight photography. The number of DD nucleic acid fluorescent complexes formed post-staining were counted (fluorescent count) and in conjunction with the fluorescence signal intensity (DD nucleic acid complex accumulation) used to estimate the level of nucleic acid persistence on substrates. Touch deposits have shown to be the most persistent deposit with strong adhesion capabilities on both substrate verities. Saliva displayed a higher persistence than semen and/or blood. Semen displayed a high collection efficiency as well as a high fluorescence signal intensity. Blood displayed a low persistence on both substrates with a superior collection efficiency that may also indicate a higher probability to become dislodged from surfaces given a particular activity. Our research has shown that the persistence and recovery of biological deposits is not only measurable but more importantly, may have the potential to be estimated, as such, may build an understanding that can provide valuable guidance for collection efficiency evaluations, and the assessing of the probability of particular profiles, given alternate propositions of means of transfer occurring.

How the physicochemical substrate properties can influence the deposition of blood and seminal deposits.

A comprehensive investigation into the impact of the physical and chemical variables of a substrate on the deposition was conducted to aid in the estimation of the subsequent transfer probabilities of blood and semen. The study focussed on surface roughness, topography, surface free energy (SFE), wettability, and the capacity for protein adsorption. Conjointly, evaluations of the physical and chemical characteristics of blood and seminal deposits were conducted, to assess the fluid dynamics of these non-Newtonian fluids and their adhesion potential to aluminium and polypropylene. A linear range of surface roughness parameters (0.5 - 3.5 µm) were assessed for their impact on the deposit deposition spread and adhesion height, to gather insight into the change in fluid dynamics of non-Newtonian fluids. Blood has shown to produce a uniform adhesion coverage on aluminium across all roughness categories while blood deposited on polypropylene exhibited a strong hydrophobic response from a surface roughness of 2.0 µm and beyond. Interestingly, the deposition height of blood resulted in near identical values, whether deposited onto the hydrophobic polypropylene or the hydrophilic aluminium substrate, illustrating the potential influence of a heightened fibrinogen adsorption effect. Semen deposited on aluminium resulted in concentrated localised deposition regions after reaching a surface roughness of 2.0 µm, highlighting the development of crystal formations afforded by the sodium ion concentration in the seminal fluid. The semen deposited on polypropylene conformed to the substrate contours producing a deposition film that was smoother than the substrate itself, underlining the effects of thixotropic fluid dynamics. Variables identified here establish the complexity observed for non-Newtonian fluids, and the effect protein adsorption may have on the deposition behaviour of blood and seminal deposits and inform questions in relation to the adhesion strength of said deposits and their ability to dislodge (becoming detached upon the application of an external force) from the substrate surface during a potential transfer event.

3-Methoxy-2-phenylimidazo[1,2-b]pyridazines highly active against Mycobacterium tuberculosis and Mycobacterium marinum.

A series of 3-methoxy-2-phenylimidazo[1,2-b]pyridazine derivatives which were highly active against autoluminescent Mycobacterium tuberculosis (Mtb) and Mycobacterium marinum (Mm) in an in vitro assay were identified. SAR analysis showed that the most active compounds, which included a phenyl group bearing fluoro substituent(s) at C2, a methoxy function at C3, and a benzyl-heteroatom moiety at C6, exhibited in vitro MIC90 values generally around 0.63-1.26 µM against Mtb and Mm. However, these compounds were inactive against Mtb in vivo (mice), and investigations revealed very short metabolic half-lives (<10 min) when incubated with mouse liver microsomes. Multiple observations of side products produced from oxidative cleavage of the imidazole moiety during the chemical synthesis work suggested that this is a likely metabolic pathway leading to the lack of observed activity in vivo.

How changes to the substrate's physical characteristics can influence the deposition of touch and salivary deposits.

An in-depth study into the physical substrate characteristics such as substrate surface roughness, topography, and physicochemical characteristics like wettability and surface free energy (SFE) was conducted to investigate the impact on the deposition and adherence of touch and salivary deposits on aluminium and polypropylene. A robust protocol was established to generate a set of substrates with a controlled linear surface roughness range (0.5-3.5 µm) in order to identify the impact of surface roughness on DNA transfer, persistence, prevalence, and recovery (DNA-TPPR). The polypropylene substrate was shown to produce fibres when artificially roughened, becoming more prominent at a higher surface roughness range, and has shown to have a direct impact on the distribution of salivary and touch deposits. At the low to moderate surface roughness range 0.5-2.0 µm, salivary and touch deposits have generally shown to follow the topographical features of the substrate they were deposited on, before a plateau of the surface roughness measure on the deposit was observed, indicating that a saturation point was reached and the grooves in the substrate were beginning to fill. Touch deposits have shown to maintain a consistent deposition height pre-surface roughness threshold, irrespective of substrate surface roughness while the deposition height of salivary deposits was heavily influenced by substrate surface roughness and topography. The substrate SFE, wettability, hydrophobicity, and the surface tension of the deposit was shown to drive the adhesion properties of the saliva and touch deposits on the respective substrates, and it was observed that this may be of importance for the improvement of the current DNA-TPPR understanding, DNA sampling protocols, and DNA transfer considerations within casework.

"Technical Note:" Optimisation of Diamond™ Nucleic Acid Dye preparation, application, and visualisation, for latent DNA detection.

A targeted sampling approach of latent DNA, deposited when a person makes contact with a surface, can prove challenging during crime scene or evidence processing, with the sampling of latent DNA often relying on the expert judgement from crime scene officers and forensic examiners. As such, the ability to use the quick and robust screening tool Diamond™ Nucleic Acid Dye (DD) was explored, with a focus on the visualisation of latent DNA on non-porous substrates, namely polypropylene, acrylic, aluminium, PVC composite material, glass, and crystalline silicon. The application of DD was performed according to methods reported in literature, where 10 µL of the dye solution (20-fold dilution of DD in 75% EtOH) was applied onto a variety of non-porous substrates via a micropipette and then subsequently visualised using a portable fluorescence microscope. It was discovered that there was scope for improvement in the reported methods due to the observation of crystal formations on all test substrates upon drying of the DD, resulting in the impaired visualisation of latent DNA and fingerprint detail. Thus, changes to the EtOH water ratio of the dye solution, and changes to the mode of dye application from a micropipette to a spray application, were explored to improve the drying time of the dye and mitigate the formation of crystals. While changes to the EtOH water ratio did not improve the overall drying time, the mode of dye application enhanced visualisation, with a spray application eliminating the formation of crystals no matter the EtOH water ratio. Visualisation with a portable Dino-Lite and Zeiss Widefield fluorescence microscope were also explored, with the Zeiss Widefield fluorescence microscope proving to be useful in whole print imaging and a more efficient imaging tool in a laboratory setting.

Intracardiac thrombosis and embolism in patients with cardiac amyloidosis.

BACKGROUND: Patients with primary amyloidosis (AL type) have a poor prognosis, in part due to frequent cardiac involvement. Although intracardiac thrombus has been reported in anecdotal cases, neither its frequency nor its role in causing mortality is known. Furthermore, the clinical and echocardiographic variables that may be associated with thromboembolism in cardiac amyloidosis have not been defined. METHODS AND RESULTS: A total of 116 autopsy or explanted cases of cardiac amyloidosis (55 AL and 61 other type) were identified in the Mayo Clinic. Forty-six fatal nonamyloid trauma cases served as controls. Each heart was examined for intracardiac thrombus. The cause of death was determined from autopsy and clinical notes. Intracardiac thrombosis was identified in 38 hearts (33%). Twenty-three had 1 thrombus, whereas 15 had 2 to 5 thrombi. Although subjects in the AL group were younger and had less atrial fibrillation than those with other types of amyloidosis, the AL group had significantly more intracardiac thrombus (51% versus 16%, P<0.001) and more fatal embolic events (26% versus 8%, P<0.03). Control hearts had no intracardiac thrombus. The presence of both atrial fibrillation and AL was associated with an extremely high risk for thromboembolism (odds ratio 55.0 [95% confidence interval 8.1 to 1131.4]). By multivariate analysis, AL type (odds ratio 8.4 [95% confidence interval 1.8 to 51.2]) and left ventricular diastolic dysfunction (odds ratio 12.2 [95% confidence interval 2.7 to 72.7]) were independently associated with thromboembolism. CONCLUSIONS: A high frequency of intracardiac thrombosis was present in cardiac amyloidosis. Furthermore, thromboembolism caused significant fatality. Several risk factors for thromboembolism were identified. Early screening, especially in high-risk patients, and early anticoagulation might reduce morbidity and mortality.

Regulatory role of dynamin-2 in VEGFR-2/KDR-mediated endothelial signaling.

Vascular endothelial growth factor receptor-2 (VEGFR-2, also known as KDR) is a receptor tyrosine kinase (RTK) regulating mitogenic, chemotactic, permeability, and survival signals in vascular endothelial cells (EC) in response to its ligand, vascular permeability factor/VEGF (VPF/VEGF), arguably the most important angiogenic cytokine. However, the compartmentalization of KDR in EC and the mechanisms regulating this process have not been well defined. Here, we demonstrate that KDR is present on the plasma membrane, on endosomes, and in the perinuclear region of EC and colocalizes with early endosomal antigen (EEA1), caveolin-1, and dynamin-2, a signal transducing GTPase involved in receptor endocytosis. Furthermore, we also observed that dynamin-2 coimmunoprecipitates with KDR and is required for EC signaling/survival. Interestingly, EC overexpressing a mutant form of dynamin deficient in GTP binding (K44A) caused a selective inhibition in KDR protein level and endosomal vesicle formation and induced cell cycle arrest by inducing p21. Taken together, our findings suggest that dynamin-2 regulates KDR expression and function and hence plays an important role in VPF/VEGF mediated angiogenesis.